Streptokinase chemically bonded to a carbohydrate matrix

ABSTRACT

One part by weight of streptokinase is chemically bonded to 1 to 500 parts by weight of a carbohydrate, to form a material which has improved stability compared with free streptokinase.

O United States Patent [151 3,639,213 Ginger et al. Feb. 1, 1972 [54]STREPTOKINASE CHEMICALLY 3,255,094 6/1966 Mather et al 195/66 BONDED ToA CARBOHYDRATE FOREIGN PATENTS OR APPLIC TIO MATRIX A n lnvemorszLeonard George g Genview; 1,577,571 8/1969 France ..l95/63 AdalineNicoles Mather, Evanston, both of ()TH R PUBLICATIONS lll. Axen et al.,Nature, April 23, 1966 pages 367- 369 Asslgneei Baxter Laboratories,Morton Grove, Silman et al., Annual Review of Biochemistry Vol. 35 pages873- 907 (pages 373- 886 relied on), 1966 Part 11. d: 1 [22] me Dec 969Primary Examiner-Lionel M. Shapiro [21] Appl. N0.: 881,632Attorney-Walter C. Kehm and W. Garrettson Ellis [52] US. Cl ..195/63,195/68, 424/94,

195 IDIG H [57] ABSTRACT [51] Int. Cl ..C07g 7/02, A6 1 k 19/00 One partby weight of streptokinase is chemically bonded to 1 [58] Field ofSearch ..l95/63 P, 68, 66 B to 500 parts by weight of a carbohydrate, toform a material which has improved stability compared with free [56]References Cited streptokinase.

UNITED STATES PATENTS 16 Claims, No Drawings STREPTOKINASE CHEMICALLYBONDED TO A CARBOHYDRATE MATRIX Streptokinase is effective to dissolvevarious types of blood clots both within and outside of the body.However, a number of difficulties are encountered in the use of theenzyme in therapeutic treatment. Antibodies against streptokinase arepresent in the blood of essentially all human beings, but the antibodylevel or titer varies widely among individuals. in order for treatmentwith streptokinase to be effective, the blood of each patient must betitrated to determine the proper initial dose of enzyme sufficient tonullify the antibodies present in the blood and to provide the properlevel of streptokinase enzyme to function as desired, while avoiding thedeleterious effects of an excessive dose of streptokinase.

Further, during treatment, the patient develops an increasedconcentration of antibodies to the enzyme. Hence, a subsequent treatmentwith streptokinase at a later date may require an increasedstreptokinase dosage. Also, a subsequent dose may be dangerous since thepatient can become sensitized to the enzyme, and may thus undergo asevere allergic reaction.

Additionally, doses of the enzyme must be adequate in the first place inorder to effectively dissolve blood clots, and they must be administeredrepeatedly or continuously, generally by intravenous drip, since theenzyme is metabolized by the body in a relatively short period of time.

Accordingly, there is a need for a substance having a streptokinaselikeactivity, yet which has increased stability in the body and which alsois less subject to neutralization by the antibodies present in the bloodstream to make possible a reduction in the amount and frequency of thedosage. Along with such reduced dosages, the danger of allergic reactionand other side effects is reduced.

DESCRIPTION OF THE INVENTION The composition of this applicationcomprises 1 part by weight of streptokinase, chemically bonded to from 1to 500 parts by weight of a carbohydrate support medium. This materialexhibits a streptokinaselike clot dissolving activity, yet it hasimproved stability in the blood stream, and is less subject toinactivation by blood antibodies than is free streptokinase. Because ofthis fact, it becomes frequently unnecessary to titrate the individualpatients blood, so that a standardized dose can be more easily utilizedto achieve the dissolution of blood clots, emboli and the like.

Preferably, streptokinase is covalently bonded to the carbohydratesupport medium with from l to 200 parts of the support medium beingpresent for each part by weight of streptokinase. It is also preferredfor the carbohydrate support medium used to be water dispersible orwater soluble so that the chemically bonded product is able to formcolloidal solutions having a particle size of less than about 2 microns.Such colloidal solutions of streptokinase chemically bonded to acarbohydrate can be directly injected, and can circulate freely in theblood without causing serious side effects due to blockage ofcapillaries and small blood vessels. In this manner, the chemicallybonded streptokinase is brought into contact with the clot to act on it.

streptokinase is produced by many strains of hemolytic streptococci,including those of Group A and Lancefield Group C streptococci. Theselected streptococci can be conventionally cultured, and the crudestreptokinase isolated in a conventional manner. Pyrogenic materials canbe removed from the streptokinase preparation, for example, by utilizingthe teachings of the Mather et al. US. Pat. No. 3,255,094.

The support media used herein may constitute carbohydrates such ascellulose, dextran, starch, dextrins, or other polysaccharides,preferably having a molecular weight of about 70,000 to about 500,000.Carbohydrate derivatives are also included in the term carbohydrate,including alkali metal containing derivatives, carbohydrate-containingpolymers such as copolymcrs of sucrose and epichlorohydrin,

carbohydrates etherified with aminoalkyl groups such as aminoethyl, andcarboxyl-containing derivatives such as carboxyethylcellulose,carboxymethyldexltran, and other carboxyalkylcarbohydrates such ascarboxypropyldextran and the like. Typically, the carbohydrate supportmedia are chemically modified to provide bonding sites for thestreptokinase enzyme.

A carbohydrate support medium having pending carboxylic acid groups,such as the carboxyalkylcarbohydrates, can be converted to an azide inthe manner typified in example I below. The azide is then reacteddirectly with streptokinase, generally at a low temperature betweenabout 0 C. and 10 C., to yield a covalently bonded addu-ct ofstreptokinase and the carbohydrate support medium of the formula inwhich R is the carbohydrate support medium having a bond on an oxygenatom thereof connected to the moiety within the brackets, R is saidstreptokinase having a bond connected to the moiety, typically on anamino nitrogen or a sulfhydryl sulfur atom of the streptokinase,referring to the condition of the atom of the streptokinase prior toreaction with the moiety, and n is a positive integer, preferably 1 or2.

Another technique for producing a chemical bond between a carbohydratesupport medium having pendant carboxylic acid groups and streptokinaseis to add a diorganocarbodiimide to a mixture of the support medium andstreptokinase in the manner exemplified by examples 4, 5, and 6, toyield a product in which carbonyl groups of the support mediumscarboxylic acid groups are directly bonded to the streptokinase,typically to amine nitrogen atoms thereof.

Carbohydrate support media can be covalently bonded to streptokinase asillustrated in example 3 by the use of a triazine of the formula tocovalently bond streptokinase to a carbohydrate through a moiety of theformula shown within the brackets in which R and R are as defined above,and X is a halogen, hydrogen, or monovalent hydrocarbon radical of nomore than about four carbon atoms. When X is a halogen, it also can bereplaced with R or R' group.

Likewise, carbohydrate support media can be chemically bonded tostreptokinase by means of cyanogen bromide as shown in example 6. Theactivation reaction is generally run under alkaline conditions, e.g., apH of at least 7.5 and preferably above 1 I.

The compositions of this invention can be used to dissolve blood clotsand the like by direct administration of a colloidal solution to theclot site, or by passing blood, plasma, or the like through a bed of aninsoluble composition of this invention. In this latter circumstance, itis generally preferred to use larger particle sizes, generally in thevisible range. An extracorporial circuit can be arranged by looselypacking a particulate composition of this invention into a cartridge, inwhich blood plasma, whole blood, or another solution passes throughtubing into one end of the cartridge, through the par ticulatecomposition of the invention, and out of the particulate composition,generally past a filter and through an exit tubing. If desired, theblood or other fluid can be directly withdrawn from a subject and/orthen administered to the subject after treatment.

Similarly, conventional reaction columns containing the material of thisinvention can be used, or the material of this invention can beimpregnated in a matrix such as silicone rubber and incorporated intubing through which the material to be treated passes.

The following examples are offered for illustrative purposes only, andare not intended to limit the scope of the invention.

EXAMPLE 1 Ten grams of the sodium salt of carboxymethyl dextran having aweight average molecular weight of about 100,000, 15 ml. of concentratedhydrochloric acid, and 250 ml. of methanol are heated at reflux for 4hours. The solvent is removed by vacuum distillation, and the residuesuspended in 50 ml. of methanol. A 20 percent solution of hydrazine inmethanol is added and stirred until no more white precipitate is formed.The precipitate is then stirred for four hours, filtered and dried. Fivegrams of the precipitated product are then resuspended in 150 ml. of 2percent hydrochloric acid, and cooled to between and 5 C. An excess ofdilute nitrous acid solution is added slowly with constant stirring.

The resulting product is precipitated and washed with methanol. Theprecipitate consists largely of dextran having units linked to oxygenatoms of the dextran, the material being informally called dextranazide.

After careful washing, the dextran azide is redissolved in a watersolution of about 0.05 to 0.1M disodium phosphate and 0.9 weight percentsodium chloride, adjusted to about pH 7.0. The concentration of dextranazide is adjusted to about 25 to 50 mg. per ml. of the phosphate-salinebuffered solution.

Purified streptokinase is then dissolved in the above solution at alevel of about 100,000 enzyme units per ml. of solution, and allowed tostand for about 12 hours (the enzyme units being as defined by theNational Institutes of Health).

The resulting mixture is then passed through a molecular sievecomprising a material such as the cross-linked dextran sold under thename Sephadex 150 or Sephadex 200, or the commercially availablematerial Biogel P, which is a crosslinked polyacrylamide.

The resulting filtrate is a solution containing dextran which iscovalently bonded to streptokinase by a linkage or moiety shown withinthe brackets of the formula 0 niomlig rn' in which R is dextran, bondedto the linkage through an oxygen atom of the dextran,and R is thestreptokinase connected to the linkage. It is believed that the majorportion of the linkages to the streptokinase are connected theretothrough amine nitrogen atoms of the streptokinase. The freestreptokinase is absorbed by the molecular sieve, and can be removed byfurther washing.

The resulting product exhibits the capability of dissolving blood clotsand the like, while also showing growing greater stability at ambientand warm temperatures when compared with free streptokinase.

Alternatively, free streptokinase can be separated from the covalentlybonded dextran-streptokinase composition by precipitation of thefreestreptokinase with ammonium sulfate, and then removing of theprecipitate by filtering or centrifuging. Excess salt and other ionicmaterials can be removed'from the dextrau-streptokinase solution bydialysis or in any other conventional manner.

EXAMPLE 2 Equivalent results are obtained when the experiment of examplel is repeated using carboxyethyldextran having a weight averagemolecular weight of about 300,000 in substitution for the sodium salt ofcarboxymethyldextran.

Furthermore, after the dextran azide is prepared from the above andfiltered, good results are obtained by dissolving the dextran azideprepared above to saturation in a 0.05 M solution of tris-(hydroxymethyl)aminomethane buffer having a pH of approximately 8.0 with the purifiedstreptokinase being then placed into the solution and allowed to standwith stirring for about 16 hours.

EXAMPLE 3 A. Dextran having a molecular weight of about 200,000 isdissolved in a saturated solution of sodium bicarbonate. Cyanuricchloride is then added in such a concentration as to provide about onemole of cyanuric chloride for each mole ofON a group present upon thedextran. The mixture is then stirred for about 1 hour at roomtemperature, filtered to remove any insoluble material, and dialyzedagainst saturated sodium bicarbonate to remove any unreacted cyanuricchloride.

Purified streptokinase is added to provide about 100,000 enzyme units(as defined above) per ml. of solution containing the dissolved cyanuricchloride-dextran reaction product, and the mixture is allowed to reactfor about 14 hours at 5 C. Any remaining free streptokinase is separatedfrom the resulting covalently bonded dextran-streptokinase by passingthe material through a molecular sieve or by precipitation of the freestreptokinase with ammonium sulfate.

The resulting material exhibits blood clot dissolving activity, and hasincreased stability with respect to free streptokinase.

B. Generally equivalent results to the above are obtained when thedextran is replaced with a sucrose-containing polymer (Ficoll, acopolymer of sucrose and epichlorohydrin, manufactured by Pharmacia ofUppsala, Sweden) having a weight average molecular weight of 400,000, orwhen cyanuric chloride is replaced with any of compounds A through Gbelow, each of the formula in which X represents the respective radicalsshown below:

bromide methyl hydrogen isopropyl n-butyl iodide allyl EXAMPLE 4Approximately 30 to 50 mg. of carboxymethyl cellulose are weighed into a25 ml. Erlenmeyer Flask. Dilute hydrochloric acid is added in a dropwisemanner to adjust the pH to between 4 and 5. From 3 to 5mg. ofdicyclohexylcarbodii- Compound A mide, and 0.2 to 0.5 mole oftetrahydrofuran (as solubilizing in which R is cellulose bonded to thelinkage through a free oxygen atom of the cellulose, and R isstreptokinase bonded to the linkage. It is believed that the majorportion of the linkages to the streptokinase are connected theretothrough amine nitrogen atoms of the streptokinase. The product showsclot decomposing activity.

Equivalent results to the above are obtained whencarboxymethyl-cellulose is replaced with a carboxymethylated copolymeror sucrose and epichlorohydrin having a weight average molecular weightof about 400,000.

EXAMPLE 5 Generally equivalent results to those obtained in example 4 Iare obtained upon substitution of dicyclohexylcarbodiimide by eitherl-cyclohexyl-3-(2-morpholinyl)-4-ethylcarbodiimidemethyl-p-toluene-sulfonate or 4-morpholinodimethylaminopropylcarbodiimide, in the absence oftetrahydrofuran.

EXAMPLE 6 EXAMPLE 7 Two grams of cyanogen bromide are dissolved in 50ml. of distilled water adjusted to a pH of l 1.5 by addition of aqueoussodium hydroxide solution. Two grams of dextran having a weight averagemolecular weight of 500,000 are added, and the suspension ismagnetically stirred for approximately 30 minutes. The dextran isprecipitated by addition of 50 percent ethyl alcohol, centrifuged, andwashed with absolute ethanol. Excess ethanol is removed under vacuum.

Five hundred mg. of the resulting material, the structure of which isuncertain at the present time are dissolved in 5 ml. of 0.10 M sodiumphosphate buffer, pH 7.5, and 5 mg. of streptokinase are added. Themixture is stirred for sixteen hours while the temperature is maintainedat 4 C.

The resulting material comprises dextran chemically bonded tostreptokinase, and has streptokinase clot dissolving activity.

Equivalent results are obtained upon substituting a commerciallyavailable copolymer of sucrose and epichlorohydrin for dextran in thisexperiment.

What is claimed is:

l. A composition of matter which comprises one part by weight ofstreptokinase, covalently bonded to from l to 500 parts by weight of adextran support medium having a molecular weight of about 70,000 to500,000, said composition of matter being capable of forming colloidalsolutions having a particular size ofless than about 2 microns.

2. The composition of claim 1 in which said dextran support medium ismodified with carboxyalkyl groups.

3. The composition of claim 1 in which from 10 to 200 parts by weight ofsaid dextran support medium are present per part by weight ofstreptokinase.

4. The composition of claim 1 in which said streptokinase is bonded to adextran support medium by at least one moiety shown within the bracketsof the formula in which R is said dextran support medium having a bondon an oxygen atom thereof connected to said moiety, R is saidstreptokinase having a bond connected to said moiety on an atom thereofand n is a positive integer.

5. The composition of claim 4 in which from l0 to 200 parts by weight ofsaid support medium are present per part by weight of streptokinase.

6. The composition ofclaim 5 in which n has a value of one to two.

7. the composition of claim 6 in which said support medium is acarboxylakyl dextran.

8. The composition of claim 1 in which said streptokinase is bonded to adextran support medium by at least one moiety shown within the bracketsof the formula I N- o R-C N in which R is said dextran support mediumhaving a bond connected to said moiety on an oxygen atom thereof, R issaid streptokinase having a bond connected to said moiety, and X isselected from the group consisting of monovalent hydrocarbon radicals ofno more than 4 carbon atoms, hydrogen, halogen atoms, and R and Rgroups.

9. The composition of claim 8 in which from 10 to 200 parts by weight ofsaid dextran support medium are present per part by weight ofstreptokinase.

10. The composition ofclaim 9 in which X is chlorine.

11. The process of contacting a dextran with cyanogen bromide underalkaline conditions to form a reaction product therebetween, andthereafter contacting one part by weight of streptokinase with from 1 to500 parts by weight of said reaction product to form a chemically bondedwater soluble adduct comprising said dextran and streptokinase.

12. The chemically bonded adduct prepared by the process of claim ll.

13. The composition of claim 12 produced by contacting from 10 to 200parts by weight of said reaction product with one part by weight ofstreptokinase.

14. The composition of claim 13 in which said alkaline conditions usedfor preparing said reaction product constitute a pH of at least ll.

15. The process of contacting from I to 500 parts by weight of adextran, having pendant groups of the formula bonded to oxygen atoms ofsaid carbohydrate, with one part by weight of streptokinase to form awater soluble covalently bonded adduct of said dextran andstreptokinase, in which n is a positive integer.

16. The process of contacting (1) from 1 to 500 parts by weight of thereaction product of a dextran and a compound of the formula in which Xis selected from the group consisting of halogen, hydrogen, andmonovalent hydrocarbon radicals of no more than 4 carbon atoms with (2)one part by weight of streptokinase to form a water soluble covalentlybonded adduct of said dextran and streptokinase.

UNITED STATES PATENT OFFTQE CERTIFICATE OF CoRREoiN 3,633213 I DatedFebruary 1, 1972 Patent No.

Invent0r(s) Leonard George Gi 31 It is certified that error appears inthe above-identified patent and that said Letters Patentare herebycorrected as shown below:

Column 5, line 62,. "particular" should read particle Signed and sealedthis 24th day of October 1972.

- (SEAL) Attest:

EDWARD M.FLETCHER,JR. 7 ROBERT GOTTSCHALK Attesting Officer Commissionerof Patents 'ORM P0-105O (10-69) USCOMM-DC 60376-P69 U5. GOVERNMENTPRINTING OFFICE: I969 O366-33

2. The composition of claim 1 in which said dextran support medium ismodified with carboxyalkyl groups.
 3. The composition of claim 1 inwhich from 10 to 200 parts by weight of said dextran support medium arepresent per part by weight of streptokinase.
 4. The composition of claim1 in which said streptokinase is bonded to a dextran support medium byat least one moiety shown within the brackets of the formula in which Ris said dextran support medium having a bond on an oxygen atom thereofconnected to said moiety, R'' is said streptokinase having a bondconnected to said moiety on an atom thereof and n is a positive integer.5. The composition of claim 4 in which from 10 to 200 parts by weight ofsaid support medium are present per part by weight of streptokinase. 6.The composition of claim 5 in which n has a value of one to two.
 7. thecomposition of claim 6 in which said support medium is a carboxylakyldextran.
 8. The composition of claim 1 in which said streptokinase isbonded to a dextran support medium by at least one moiety shown withinthe brackets of the formula
 9. The composition of claim 8 in which from10 to 200 parts bY weight of said dextran support medium are present perpart by weight of streptokinase.
 10. The composition of claim 9 in whichX is chlorine.
 11. The process of contacting a dextran with cyanogenbromide under alkaline conditions to form a reaction producttherebetween, and thereafter contacting one part by weight ofstreptokinase with from 1 to 500 parts by weight of said reactionproduct to form a chemically bonded water soluble adduct comprising saiddextran and streptokinase.
 12. The chemically bonded adduct prepared bythe process of claim
 11. 13. The composition of claim 12 produced bycontacting from 10 to 200 parts by weight of said reaction product withone part by weight of streptokinase.
 14. The composition of claim 13 inwhich said alkaline conditions used for preparing said reaction productconstitute a pH of at least
 11. 15. The process of contacting from 1 to500 parts by weight of a dextran, having pendant groups of the formulabonded to oxygen atoms of said carbohydrate, with one part by weight ofstreptokinase to form a water soluble covalently bonded adduct of saiddextran and streptokinase, in which n is a positive integer.
 16. Theprocess of contacting (1) from 1 to 500 parts by weight of the reactionproduct of a dextran and a compound of the formula in which X isselected from the group consisting of halogen, hydrogen, and monovalenthydrocarbon radicals of no more than 4 carbon atoms with (2) one part byweight of streptokinase to form a water soluble covalently bonded adductof said dextran and streptokinase.